Human DNMT2(DNA Methyltransferase 2)ELISA Kit (HUES03428)

SKU:
HUES03428
€539

Description

ELISA Kit Technical ManualMSDS

Human DNMT2(DNA Methyltransferase 2)ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human DNMT2 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human DNMT2 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human DNMT2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human DNMT2. The concentration of Human DNMT2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human DNMT2 in samples. No significant cross-reactivity or interference between Human DNMT2 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
O.D Average Corrected
50002.512
2.546
2.5292.447
25001.645
1.681
1.6631.581
12501.038
1.018
1.0280.946
6250.521
0.527
0.5240.442
312.50.289
0.261
0.2750.193
156.250.198
0.184
0.1910.109
78.130.127
0.147
0.1370.055
00.082
0.082
0.082--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human DNMT2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human DNMT2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)268.58547.961928.18259.80573.672007.74
Standard deviation18.2923.2378.6714.1329.09103.80
C V (%)6.814.244.085.445.075.17

Recovery

The recovery of Human DNMT2 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)92-10899
EDTA plasma (n=5)84-9990
Cell culture media (n=5)91-10597

Linearity

Samples were spiked with high concentrations of Human DNMT2 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)91-10487-10084-97
Average (%)969291
1:4Range (%)92-10485-9685-98
Average (%)978991
1:8Range (%)93-10781-9589-102
Average (%)998695
1:16Range (%)92-10483-9585-100
Average (%)998992

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human DNMT2(DNA Methyltransferase 2)ELISA Kit (HUES03428) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human DNMT2(DNA Methyltransferase 2)ELISA Kit (HUES03428) Protein Information

UniProt Protein Function:TRDMT1: Specifically methylates cytosine 38 in the anticodon loop of tRNA(Asp). Belongs to the C5-methyltransferase family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Amino Acid Metabolism - cysteine and methionine; EC 2.1.1.204; Methyltransferase; Methyltransferase, tRNA

Chromosomal Location of Human Ortholog: 10p13

Cellular Component: cytoplasm; nucleoplasm

Molecular Function:tRNA (cytosine-5-)-methyltransferase activity; tRNA methyltransferase activity

Biological Process: tRNA methylation; tRNA modification

NCBI Summary:This gene encodes a protein responsible for the methylation of aspartic acid transfer RNA, specifically at the cytosine-38 residue in the anticodon loop. This enzyme also possesses residual DNA-(cytosine-C5) methyltransferase activity. While similar in sequence and structure to DNA cytosine methyltransferases, this gene is distinct and highly conserved in its function among taxa. [provided by RefSeq, Jun 2010]
UniProt Code:O14717
NCBI GenInfo Identifier:17374834
NCBI Gene ID:1787
NCBI Accession:O14717.1
UniProt Secondary Accession:O14717,O43669, Q86WW6, B0YJ02, B0YJ03, B0YJ07, B0YJ08
UniProt Related Accession:O14717
Molecular Weight:45kDa
NCBI Full Name:tRNA (cytosine(38)-C(5))-methyltransferase
NCBI Synonym Full Names:tRNA aspartic acid methyltransferase 1
NCBI Official Symbol:TRDMT1
NCBI Official Synonym Symbols:DMNT2; DNMT2; PUMET; RNMT1; MHSAIIP
NCBI Protein Information:tRNA (cytosine(38)-C(5))-methyltransferase
UniProt Protein Name:tRNA (cytosine(38)-C(5))-methyltransferase
UniProt Synonym Protein Names:DNA (cytosine-5)-methyltransferase-like protein 2; Dnmt2; DNA methyltransferase homolog HsaIIP; DNA MTase homolog HsaIIP; M.HsaIIP; PuMet
Protein Family:tRNA
UniProt Gene Name:TRDMT1
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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